The present invention relates to a method and an apparatus for classifying B-lymphocytes in a blood sample. The leukocytes in a peripheral blood of a healthy human being can be classified into lymphocytes, monocytes, neutrophils, eosinophils, and basophils. The lymphocytes are further roughly classified into three types called subsets (T-lymphocytes, B-lymphocytes, and NK-lymphocytes). The B-lymphocytes are lymphocytes derived from bone marrow and have immunoglobulin on the surface thereof. Further, the B-lymphocytes play a very important role in an immunity function of a human being such as production of antibodies by being differentiated into plasma cells. Usually, a B-lymphocyte ratio (%) is used to represent the ratio occupied by the B-lymphocytes in the total lymphocytes.
Since the B-lymphocytes are strongly involved in immunity mechanism, the increase or decrease thereof is directly linked to capturing the change in the immunity function. At ordinary times, the B-lymphocytes are present at a constant ratio; however, the ratio may change in accordance with the presence of a disease. Therefore, measurement of a B-lymphocyte ratio is useful in obtaining information on the presence of a disease.
Examples of the diseases showing increase in the B-lymphocytes include multiple myeloma, B-lymphocyte type chronic lymphocytic leukemia, infectious mononucleosis, Burkitt's lymphoma, and others. Examples of the diseases showing decrease in the B-lymphocytes include AIDS, progressive cancer, Hodgkin's lymphoma, agammaglobulinemia, measles, chicken pox, herpes, and others.
As a technique for classifying and counting lymphocytes, manual methods have often been used, such as the rosette method in which, by using the fact that sheep erythrocytes sensitized with a complement bind to B-lymphocytes, the number thereof is counted while the smear sample thereof is being observed with an optical microscope, or the surface immunity fluorescence method in which a smear sample of blood is labeled with a fluorescence-labeled antibody and the number of lymphocytes is counted while the smear sample is being observed with a fluorescence microscope. However, in recent years, a flow system is generally used in which blood cells labeled with a fluorescence-labeled antibody are let to flow in a liquid and the blood cells are classified and counted by performing signal processing on signals obtained from individual blood cells.
In a flow system, blood cells are let to flow in a liquid, and a signal is detected for each blood cell (for example, a signal based on the difference of optical characteristics). In doing this, by adding an antibody corresponding to the cells that should be detected after labeling the antibody with a fluorescent substance, only the cells that should be detected emit fluorescence signals, and information on the desired cells can be obtained by detecting these signals (See U.S. Pat. No. 4,284,412).
Antibodies have a property of binding only to a specific substance, and are known to have an extremely high specificity thereof. At present, substances (such as protein) appearing on specific cells are specified and, by using the antibodies that are prepared to bind to this substance, one can perform a fine classification of cells. Antibodies (for example, anti-CD19, anti-CD20 antibodies) that are used for classifying and counting B-lymphocytes are prepared to bind to a substance that appears only on B-lymphocytes.
The fluorescence-labeled antibodies to be used require an extremely high technique and time for preparation thereof, and also are very expensive though used in a slight amount.
Further, The flow system using the fluorescence-labeled antibodies requires a complex procedure for letting a slight amount of cells bind to a slight amount of fluorescence-labeled antibodies without loss for measurement, and requires repetition of short-time operations and a period of time for waiting.